Current Medicinal Chemistry, 2008, 15, 1249-1256 1249 Of Stem Cells and Gametes: Similarities and Differences Bernard A.J. Roelen*,1 and Susana M. Chuva de Sousa Lopes2 1Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands; 2Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Abstract: Fusion of a mammalian sperm cell with an oocyte will lead to the formation of a new organism. As this new organism devel- ops, the cells that construct the organism gradually lose developmental competence and become differentiated, a process which is in part mediated via epigenetic modifications. These mechanisms include DNA methylation, histone tail modifications and association with Polycomb and Trithorax proteins. Several cells within the organism must however maintain or regain developmental competence while they are highly specialized. These are the primordial germ cells that form the gametes; the oocytes and sperm cells. In this review differ- ent epigenetic modifying mechanisms will be discussed as they occur in developing embryos. In addition, aspects of nuclear reprogram- ming that are likely to occur via removal of epigenetic modifications are important, and several epigenetic removal mechanisms are in- deed also active in developing germ cells. In vivo, a pluripotent cell has the capacity to form gametes, but in vitro terminal gametogenesis has proven to be difficult. Although de- velopment of pluripotent cells to cells with the characteristics of early germ cells has been unequivocally demonstrated, creating the cor- rect culture milieu that enables further maturation of these cells has as yet been futile. Keywords: Embryo, primordial germ cell, epigenetics, pluripotency, ES cell, methylation. CELLULAR POTENCY Interestingly, immature oocytes do not support reprogramming of somatic nuclei. Different pluripotent cells lines can be derived by After an oocyte has fused with a sperm cell, a new organism long term culture of distinct cell populations: embryonic stem cells will be formed. During the development of this organism, the cells (ES cells) are derived from the inner cell mass of an embryonic day that construct the embryo will gradually lose developmental poten- (E)3.5 mouse blastocyst, epiblast stem cells (EpiSCs) from an E5.5- tial and gain more specialized functions. In this respect, differentia- E6.5 mouse epiblast, embryonal carcinoma cells (EC cells) from a tion equals a loss in cellular potency. One group of cells however mouse epiblast or germ cell-induced teratomas, embryonic germ has to maintain the capacity to form a new organism. This group cells (EG cells) from E8.5-E13.5 murine PGCs and adult sper- encompasses the primordial germ cells (PGCs) that will give rise to matogonial stem cells (SSCs) from neonatal and adult spermatogo- the gametes later in development. Germ cells are unique cells since nia [5-12]. they are responsible for the continuity of genetic information across generations. They therefore have to maintain a certain level of cel- Exactly how pluripotency is regulated and how germ cells are lular potency. In vertebrates, PGCs belong to the first embryonic prevented from differentiation into somatic cells is unknown, but lineage to be segregated, long before the gonads are recognizable. our biochemical understanding of these processes has increased This specification event occurs outside the embryo at the border considerably in the last few years. It has become clear that mecha- between the extraembryonic and embryonic region so that the PGCs nisms that play an important role in maintaining potency of (pri- can escape differentiation signals. Thereafter, the PGCs follow a mordial) germ cells are also active in stem cells, but that simultane- complex migratory pattern to finally colonize the gonads [1]. ously many differences exist. Once in the gonads, male PGCs enter mitotic arrest (pre- EPIGENETIC REGULATION AS A DETERMINANT OF spermatogonia cells) whereas female PGCs enter meiosis (oogo- CELLULAR POTENCY nia). In males, the pre-spermatogonia cells start proliferating only after birth and give rise to spermatogonia which either self-renew or Although the genome in almost all cells of an organism remains differentiate to cells that enter meiosis forming haploid sperm cells. intact, in differentiating cells it is partitioned into active and quies- Spermatogonia are therefore considered an adult stem cell popula- cent genes. This can be achieved by epigenetic modification of the tion. The oogonia, on the other hand, undergo meiosis synchro- chromatin (DNA and histones), which determines the accessibility nously during embryonic development and arrest in the diplotene of genes in a heritable fashion. There are several mechanisms by stage at birth. After puberty, oocytes periodically mature, resuming which chromatin can be epigenetically programmed, including meiosis to arrest again at metaphase II after ovulation and complete DNA methylation at cytosine-phosphate-guanine (CpG) dinucleo- meiosis only after fertilization [2]. Although the sperm and the tides, covalent post-translational modification of the histone pro- oocyte are highly specialized cells, they also must maintain or re- teins or the use of histone variants in the nucleosome and the asso- gain a level of cellular naïveté. ciation with ATP-dependent complexes [13]. Most differentiated cells retain an intact genome without altera- tions in their DNA sequences, but epigenetic mechanisms modify METHYLATING THE DNA accessibility to parts of the genome leading to cell fate determina- tion and cell function [3]. However, when the nucleus of a differen- Methylation of DNA at CpG sequences is mostly associated tiated cell is introduced into the cytoplasm of a mature oocyte, that with transcriptional repression. It is widely used to ensure the si- somatic nucleus can be reprogrammed to a totipotent state and the lencing of repetitive DNA (constitutive heterochromatin), including resulting cell can give rise to a new organism genetically identical satellite DNA and parasitic transposable elements; the transient to the somatic cell donor nucleus, a clone [4]. silencing of specific genes or whole chromosomes (facultative het- erochromatin); and the monoallelic expression of imprinted genes. It consists of the addition of a methyl group to the 5-position of a cytosine in a CpG dinucleotide (Fig. 1). The CpG methylation is symmetrical, i.e. occurring on both DNA strands and it directly *Address correspondence to this author at the Department of Farm Animal Health, inhibits the binding of specific transcription factors to DNA Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands; Tel: + 31 30 2533352; Fax: +31 30 2534811; whereas it promotes the binding of methyl-CpG-binding proteins E-mail: [email protected] like MECP2, MBD1, MBD2, MBD3, MBD4 and Kaiso [13]. 0929-8673/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd. 1250 Current Medicinal Chemistry, 2008 Vol. 15, No. 13 Roelen and Lopes Stretches of CpG sequences known as CpG islands present in the [23]. Absence of DNMTs in ES cells increased telomere recombi- promoter sequences of many genes are predominantly unmethylated nation and telomere size, suggesting that DNA methylation is in- [14], whereas CpG islands in imprinted genes and genes on the volved in regulation of telomere length and therefore maintenance inactive X-chromosome are frequently methylated [14, 15]. of chromosome stability [24]. The ES mutant cells are viable but A die when induced to differentiate. Mice knockout for Dnmt1 and Cytosine Guanine Dnmt3b are embryonic lethal and mice knockout for Dnmt3a die before reaching adulthood (reviewed by [13]). Deficiency in the NH2 O methyl-CpG-binding domain (MBD) protein 3 results in embryonic NH lethality and Mbd3 knockout ES cells show a restricted potential to N differentiate [25]. It is clear that DNA methylation is of crucial N NH2 importance for reprogramming the genome in embryonic develop- N ment, gametogenesis and differentiation of ES cells. O N N O- IMPRINTING O O O P O O In viviparous mammals, and interestingly also in flowering O plants, the epigenetic changes of a selected panel of genes are in- B herited to the next generation, depending on the parental origin of the allele. The discovery of imprinting demonstrated that the mater- Methylcytosine Guanine nal and paternal genomes are functionally non-equivalent, even though they share equivalent genetic information [26, 27]. There- NH O 2 fore, embryos consisting of two fully imprinted maternal or paternal NH CH3 genomes cannot develop to term. These two kinds of embryos de- N N NH2 velop opposite phenotypic characteristics: the ones containing only N maternal genes develop better embryos and the ones containing O N N only paternal genes develop better placental tissues. Accordingly, the imprinted genes have various functions including embryonic O- O O and placental growth and suckling behaviour but why imprinting has evolved is still a matter of debate [28]. Imprinting occurs at O P O O close to 100 loci (at least in the mouse) and these appear in regional O clusters on specific chromosomes rather than being distributed Fig. (1). Methylation of cytosine in a CpG dinucleotide. A) Unmethylated evenly throughout the genome [29]. CpG dinucleotide and B) methylation of cytosine on the 5 position. Several mechanisms have been identified by which